The systematic generation and integration of high-coverage physical and transcript
maps in the chromosomal region Xq28 has facilitated the isolation of most genes
in this region, as well as the identification of genes causally associated with
diseases. We are currently analysing the developmental and tissue-specific expression
of a selection of genes in Xq28 by RNA in situ hybridisation. Functional analyses
include determination of the intracellular localisation of the corresponding
proteins by using the enhanced green fluorescent protein (EGFP) as a tag, as
well as generating antibodies and working on murine models in some cases. To
complement the functional analyses on genes in Xq28 we are also carrying out
evolutionary studies in this region. The DKC1 gene is responsible for causing
the bone marrow failure syndrome dyskeratosis congenita (DKC) and is one example
of a gene which we are characterising at the RNA expression and protein level.
The DKC1 transcript is expressed ubiquitously and very early in murine embryological
development. The protein dyskerin is highly conserved and appears to be involved
in the pseudouridylation and cleavage of pre-rRNA. Dyskerin fused to the EGFP
and expressed in mammalian cell lines localises to the nucleoplasm, the nucleoli,
and the coiled bodies which are functionally associated with the nucleoli. Mutant
constructs permitted a delineation of the sequences responsible for the nuclear
targeting and indicated that mislocalisation of dyskerin is not a mechanism
involved in the pathogenesis of DKC. Examples of genes in Xq28 being studied
at the evolutionary level are rab GDI and XAP5. The autosomal paralogues of
genes related to rab GDI and to XAP5 were isolated and mapped, and the amphioxus
and fugu orthologues of the rab GDI genes were isolated. A comparison of the
tissue-specific expression indicates that the various homologues have specialised
functions.